Immobilized nucleic acid complexes for sequence analysis

ABSTRACT

Provided are methods for sequencing a nucleic acid that include fixing a template to a surface through a template localizing moiety and sequencing the nucleic acid with a sequencing enzyme, e.g. a polymerase or exonuclease. The sequencing enzyme can optionally be exchanged with a second sequencing enzyme, which continues the sequencing of the nucleic acid. The template localizing moiety can optionally anneal with the nucleic acid and/or associate with the sequencing enzyme. Also provided are compositions comprising a nucleic acid fixed to a surface via a template localizing moiety, and a first sequencing enzyme, which can sequence the nucleic acid and optionally exchange with a second sequencing enzyme present in the composition. Compositions in which a template localizing moiety is immobilized on a surface are provided. Compositions for sequencing reactions are provided. Also provided are sequencing systems comprising reaction regions in which or near which template localizing moieties are immobilized.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/192,634, filed Sep. 19, 2008, the disclosure of which is incorporatedherein by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Nucleic acid sequence data is valuable in myriad applications inbiological research and molecular medicine, including determining thehereditary factors in disease, in developing new methods to detectdisease and guide therapy (van de Vijver et al. (2002) “Agene-expression signature as a predictor of survival in breast cancer,”New England Journal of Medicine 347: 1999-2009), and in providing arational basis for personalized medicine. Obtaining and verifyingsequence data for use in such analyses has made it necessary forsequencing technologies to undergo advancements to expand throughput,lower reagent and labor costs, and improve accuracy (See, e.g., Chan, etal. (2005) “Advances in Sequencing Technology” (Review) MutationResearch 573: 13-40, and Levene et al. (2003) “Zero Mode Waveguides forSingle Molecule Analysis at High Concentrations,” Science 299: 682-686),the disclosures of which are incorporated herein in their entireties forall purposes.

Single molecule real-time sequencing (SMRT) is a highly parallelsequencing-by-synthesis technology that permits the simultaneoussurveillance of, e.g., thousands of sequencing reactions in arrays ofmultiplexed detection volumes, e.g., zero-mode waveguides (ZMWs). (Seee.g., Levene et al. (2003) Zero-mode waveguides for single-moleculeanalysis at high concentrations, Science 299:682-686; Eid, et al. (2009)Real-Time DNA Sequencing from Single Polymerase Molecules, Science323:133-138; Published U.S. Patent Application No. 2003/0044781; andU.S. Pat. No. 6,917,726, the disclosures of which are incorporatedherein in their entireties for all purposes). Each detection volume inan array creates an illuminated visualization chamber that is smallenough to observe the template-dependent synthesis of a singlesingle-stranded DNA molecule by a single DNA polymerase.

When a particular base in the template strand is encountered by thepolymerase during the polymerization reaction, e.g., in a ZMW, theenzyme complexes with an available fluorescently labeled nucleotide ornucleotide analog and incorporates that nucleotide or nucleotide analoginto the nascent growing nucleic acid strand. During this time, thefluorophore emits fluorescent light whose color corresponds to thenucleotide's or analog's base identity. The polymerase cleaves the bondlinking the fluorophore to the nucleotide or analog during thenucleotide incorporation cycle, permitting the dye to diffuse out of thedetection volume. The signal returns to baseline, and the processrepeats.

A single molecule sequencing reaction is typically localized to adetection volume by immobilizing a DNA polymerase enzyme within orproximal to the site at which the reaction takes place. Ideally, theimmobilized polymerase retains its activity and can be used repeatedlyand continuously in multiple sequencing reactions. However, it has beenobserved that in some cases, the processivity, accuracy, and/or activityof the polymerase enzyme can decrease. In particular, in at least somecases, damage to the DNA polymerase, e.g., by exposure to optical energyduring fluorescent or chemiluminescent detection, can have a detrimentaleffect on the enzyme's activity.

Current strategies for single molecule sequencing-by-synthesis employ apolymerase that has been tethered within or proximal to a reactionregion within a detection volume, e.g., in a ZMW. What is needed in theart are new methods and compositions that can maintain the processivity,accuracy, and polymerase activity in, e.g., a single-molecule sequencingreaction, while still localizing the polymerization reaction to adefined observation volume. The invention described herein fulfillsthese and other needs, as will be apparent upon review of the following.

SUMMARY OF THE INVENTION

In certain aspects, the present invention provides methods and relatedcompositions useful for immobilizing a template nucleic acid (or“nucleic acid template”) at a reaction region. The compositions includea template localizing moiety that is covalently attached to a surface,e.g., a single molecule reaction region. The moiety can associate with atemplate nucleic acid, e.g., a DNA, RNA, or analogs or derivativesthereof, present in the composition and fix the template to the surface,e.g., localizing the nucleic acid to the surface. A sequencing enzyme,e.g., a polymerase, reverse transcriptase, exonuclease, etc., canoptionally associate with the template localizing moiety and performtemplate-directed sequencing of the template nucleic acid. In preferredembodiments, the sequencing enzyme can exchange with other sequencingenzymes present in the composition without disrupting or terminatingsequencing of the template, thus permitting, e.g., a photodamagedsequencing enzyme to exchange with a non-photodamaged sequencing enzyme.Immobilizing a nucleic acid template via a template localizing moietycan advantageously allow longer uninterrupted sequence reads in, e.g.,synthesis- or degradation-based single-molecule sequencing reactions. Incertain aspects, the present invention provides methods and relatedcompositions useful for performing template-directed synthesis of anucleic acid. In certain aspects, the invention provides methods andrelated compositions for performing exonuclease sequencing of a nucleicacid.

Thus, in a first aspect, the invention provides methods of performingtemplate-directed synthesis of a nucleic acid that include fixing atemplate nucleic acid to a solid surface through a template localizingmoiety, e.g., that topologically encircles the template. The templatelocalizing moiety can be a polymer, including but not limited to apolypeptide (e.g., other than a polymerase to be used in thetemplate-directed synthesis reaction), polynucleotide, syntheticpolymer, and combinations thereof. The methods include synthesizing anascent strand from at least a portion of the template nucleic acid witha first polymerase, exchanging the first polymerase with a secondpolymerase, and continuing synthesis of the nascent strand with thesecond polymerase. Optionally, exchanging the first polymerase caninclude exchanging a photodamaged polymerase with a polymerase that isnot photodamaged, and synthesis can optionally be continued with thesecond, non-photodamaged polymerase. Such embodiments can furthercomprise a template nucleic acid that is circular. In certain preferredembodiments the template nucleic acid is subjected to thetemplate-directed synthesis reaction multiple times with one or morepolymerases to generate a single nucleic acid strand comprising multiplecopies of a polynucleotide complementary to the template nucleic acid.

In a further aspect, the invention provides methods of performingexonuclease sequencing of a nucleic acid that include fixing a templatenucleic acid to a solid surface through a template localizing moiety,e.g. a polypeptide other than a polymerase or other polymer thattopologically encircles the template. The methods include degrading afirst strand of the template nucleic acid with a first exonuclease anddetecting the nucleotides so released, exchanging the first exonucleasewith a second exonuclease, and continuing degradative sequencing of thefirst strand with the second exonuclease. Optionally, exchanging thefirst exonuclease can include exchanging a photodamaged exonuclease withan exonuclease that is not photodamaged, and degradation can optionallybe continued with the second, non-photodamaged exonuclease.

In a related aspect, the invention provides compositions that can beused in the methods described above. The compositions include a templatenucleic acid tethered to a solid surface through a template localizingmoiety, e.g., a moiety that topologically encircles the template, and afirst sequencing enzyme capable of sequencing the template nucleic acid.The template localizing moiety can comprise a polymer (natural orsynthetic), e.g., a polypeptide, polynucleotide, synthetic polymer, andanalogs, derivatives, mimetics, and combinations thereof. In certainspecific embodiments, the template localizing moiety comprises aprotein, e.g., a hexameric helicase, a PCNA, a T4 phage gp45 protein, ora β subunit of a eubacterial DNA polymerase. In other specificembodiments, the template localizing moiety comprises a polynucleotidecomprising a nucleotide sequence complementary to a portion of thetemplate nucleic acid, and the first sequencing enzyme is a polymerasecapable of strand displacement of the polynucleotide from the templatenucleic acid. In certain embodiments, the first sequencing enzyme is afirst polymerase, e.g., capable of synthesizing a nascent strand basedon the nucleotide sequence of the template nucleic acid, and thetemplate localizing moiety permits the first polymerase to be exchangedwith a second polymerase present in the composition without terminatingtemplate-directed synthesis, e.g., the second polymerase is capable ofcontinuing the sequencing of the template nucleic acid. The polymerasecan optionally be, e.g., a DNA or RNA polymerase, e.g., a Klenowfragment, Φ29, AMV, B103, GA-1, HIV-1 PZA, Φ15, BS32, M-MLV, M2Y, Nf,G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, T4, anArcheal, an Eukaryal, or an Eubacterial polymerase, or mutations ormodified versions thereof. Optionally, the template nucleic acid may besingle-stranded or circular, and in some preferred embodiments is bothsingle-stranded and circular. Optionally, the polymerase present in thecompositions can be non-covalently attached to the template localizingmoiety.

The compositions can optionally include ATP, CTP, GTP, TTP, UTP or ITP,which can modulate the rate of polymerization in aconcentration-dependent manner, e.g., when the template localizingmoiety and the polymerase participate in a template-dependentpolymerization reaction. The compositions can optionally include one ormore fluorescently labeled nucleotides or nucleotide analogs that canphotodamage the polymerase. In some embodiments, the template localizingmoiety is not susceptible to photo-induced damage caused by the one ormore fluorescently labeled nucleotide or nucleotide analogs.

Compositions that include a template localizing moiety immobilized on aplanar surface, in a well, or in a single molecule reaction region,e.g., a zero-mode waveguide are also provided by the invention. Theimmobilized moiety can optionally comprise, e.g., a polymer (e.g.,natural or synthetic) including but not limited to a polynucleotideand/or a polypeptide, e.g., a protein other than a polymerase, such as aprocessive nuclease, a single-strand binding protein (SSBP), a helicase,a DNA repair enzyme, a DNA processivity factor, or a protein thatnon-specifically binds a double-stranded nucleic acid. The templatelocalizing moiety can optionally topologically encircle a template DNAstrand when a DNA strand is present in the composition. The templatelocalizing moiety that topologically encircles the template canoptionally comprise a PCNA, a T4 phage gp45 protein, a β subunit of aeubacterial polymerase, one or more synthetic structural units, and/or apolynucleotide, where the polynucleotide optionally comprises a portionthat is complementary to at least a portion of the template nucleicacid. In certain preferred embodiments, the template localizing moietythat topologically encircles the template comprises at least onepolynucleotide portion and at least one portion comprising syntheticstructural units, e.g., at least some of which are polyethylene glycolunits. The compositions can optionally include a template DNA, e.g., asingle-stranded DNA and/or a closed loop of DNA, which the templatelocalizing moiety can associate with and/or retain, and fix to theplanar surface, in a well, or in a single molecule reaction region,e.g., comprising a zero-mode waveguide.

Compositions in which a template localizing moiety is immobilized to aplanar surface, well, or single-molecule reaction region can optionallyinclude a sequencing enzyme, e.g., an exonuclease (e.g., T7 exonuclease,lambda exonuclease, mung bean exonuclease, ExoI, Exo III, Exo IV,ExoVII, exonuclease of Klenow fragment, exonuclease of PolI, Taqexonuclease, T4 exonuclease, etc.) or DNA polymerase (e.g., a Klenowfragment, Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE,SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.) Optionally, thesequencing enzyme can be non-covalently attached to the moiety, or itcan be covalently attached to the moiety, e.g., via a DNA polymerase'sC-terminal end. The template localizing moiety can optionally improvethe accuracy and/or processivity of the sequencing enzyme, when themoiety and the sequencing enzyme participate in a nucleic acidsequencing reaction, e.g., a sequencing-by-synthesis reaction ordegradation-based sequencing reaction. These compositions can optionallyinclude ATP, CTP, GTP, TTP, UTP or ITP, and/or one or more fluorescentlylabeled nucleotides or nucleotide analogs, as described above.

In certain embodiments, the invention provides sequencing reactions thatinclude a nucleic acid template, a synthesis initiating moiety thatcomplexes with or is integral to the template, a DNA polymerase, and atemplate localizing moiety immobilized on a substrate, e.g., a planarsurface, well, or single molecule reaction region, e.g., a zero modewaveguide. The DNA polymerase of the sequencing reaction can optionallyassociate with the immobilized template localizing moiety. Thepolymerase and the template localizing moiety can optionally benon-covalently attached. Optionally, the DNA polymerase can becovalently attached to the moiety, e.g., via the polymerase's C-terminalend.

In certain embodiments, the invention provides sequencing reactions thatinclude a nucleic acid template, a synthesis initiating moiety thatcomplexes with or is integral to the template, a DNA polymerase, atemplate localizing moiety immobilized on a substrate, which cancomprise a planar surface, a well, and/or a single molecule region,e.g., a zero-mode waveguide. In certain embodiment, the sequencingreactions provided herein further comprise a luciferase-based detectionsystem for monitoring pyrophosphate release. The DNA polymerase orcomponents of the luciferase-based detection system (e.g., luciferase,sulfurylase, etc.) can optionally associate (covalently ornon-covalently) with the immobilized template localizing moiety.

The sequencing reactions provided by the invention can optionallyinclude one or more fluorescently labeled nucleotides or nucleotideanalogs. A polymerase present in the sequencing reaction can optionallysynthesize a complementary nascent strand from at least a portion of thetemplate in a template-dependent matter, optionally incorporating one ormore fluorescently labeled nucleotides or nucleotide analog into theresulting nascent strand. In certain embodiments, the sequencingreaction comprises a pool of nucleic acid templates, and optionally, thetemplate localizing moiety (or plurality thereof) comprises apolynucleotide complementary to only one or a subset of the nucleic acidtemplates in the pool. The polymerase can be non-covalently orcovalently attached to the template localizing moiety, e.g., at aC-terminal portion of the polymerase.

In a related aspect, the invention provides sequencing systems thatinclude a reaction region, e.g., a planar surface, one or more well, orone or more single molecule reaction region, and a template localizingmoiety immobilized within or proximal to the reaction region.Optionally, the single-molecule reaction region included in the systemscan be a zero-mode waveguide. Optionally, the systems can include asequencing enzyme (e.g., a polymerase or nuclease) in the reactionregion. The template localizing moiety in the systems can optionally beconfigured to interact with a sequencing enzyme, when a sequencingenzyme is present in the reaction region. The sequencing enzyme and thetemplate localizing moiety can optionally be covalently attached ornon-covalently attached, as described above.

The systems of the invention also include a detector configured todetect a sequencing product formed in the reaction region. A sequencingproduct of the invention includes but is not limited to a newlysynthesized nucleic acid strand (“nascent strand”), releasedpyrophosphate, and nucleotides released by exonuclease degradation. Thedetector can optionally be configured to detect fluorescent light fromone or more fluorophores that is, e.g., linked to a nucleotide ornucleotide analog. The system can optionally comprise an epi fluorescentdetector.

In a further aspect, the invention provides a method of sequencing atemplate nucleic acid that includes fixing a circular template to asolid surface through a template localizing moiety, annealing anoligonucleotide primer to the template nucleic acid, initiatingtemplate-directed nascent strand synthesis by a polymerase that is notimmobilized to the solid surface, and detecting incorporations ofnucleotides into the nascent strand. A temporal sequence of theincorporations is indicative of the sequence of the nucleic acid.Optionally, the incorporations are detected by monitoring signals fromdetectable labels linked to the nucleotides as they are beingincorporated into the nascent strand, e.g., where the type of detectablelabel corresponds to the base composition of a nucleotide. Preferably,the detectable labels are removed during incorporation resulting in anascent strand that does not comprise the detectable labels. Optionally,the incorporations are detected using a luciferase-mediated detectionsystem. In certain preferred embodiments, the template localizing moietytopologically encircles the template nucleic acid. In some embodiments,the template nucleic acid is a single-stranded nucleic acid molecule.The sequencing methods can further comprise sequencing the templatenucleic acid multiple times to generate a single nascent strandcomprising multiple copies of a polynucleotide complementary to thetemplate nucleic acid. Further, in some embodiments the polymerase is aplurality of polymerase enzymes, wherein only a single polymerase enzymeis engaged in template-directed nascent strand synthesis on a singletemplate at a given time.

Those of skill in the art will appreciate that the methods provided bythe invention for sequencing of a nucleic acid, e.g., a DNA, can be usedalone or in combination with any of the compositions described herein.DNA sequencing systems that include any of the compositions describedherein are also a feature of the invention. Such systems can optionallyinclude detectors, array readers, excitation light sources, and thelike.

The present invention also provides kits that incorporate thecompositions of the invention. Such kits can include, e.g., a templatelocalizing moiety packaged in a fashion to permit its covalent bindingto a surface of interest. Alternatively, the surface bound templatelocalizing moieties can be provided as components of the kits, or thesurface can be provided with binding partners suitable to bind thetemplate localizing moieties, which are optionally packaged separately.Instructions for making or using surface bound template localizingmoieties are an optional feature of the invention.

Such kits can also optionally include additional useful reagents such asone or more nucleotide analogs, e.g., for sequencing, nucleic acidamplification, or the like. For example, the kits can include a DNApolymerase packaged in such a manner as to enable its use with thetemplate localizing moiety, a set of different nucleotide analogs of theinvention, e.g., those that are analogous to A, T, G, and C, e.g., whereone or more of the analogs comprise a detectable moiety, to permitidentification in the presence of the analogs. The kits of the inventioncan optionally include natural nucleotides, a control template, andother reagents, such as buffer solutions and/or salt solutions,including, e.g., divalent metal ions, i.e., Mg⁺⁺, Mn⁺⁺ and/or Fe⁺⁺,standard solutions, e.g., dye standards for detector calibration, etc.Such kits can optionally include various sequencing enzymes (e.g., oneor more polymerases or nucleases), and components required for detectionof a sequencing product, e.g., luciferase-based detection system. Suchkits also typically include instructions for use of the compounds andother reagents in accordance with the desired application methods, e.g.,nucleic acid sequencing, nucleic acid labeling, amplification and thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic depiction of a surface-immobilized templatelocalizing moiety fixing a template nucleic acid to the surface bytopologically encircling the template.

FIG. 2 provides a schematic depiction of a surface immobilized templatelocalizing moiety that has fixed a closed nucleic acid loop within asingle molecule reaction region.

FIG. 3 depicts a template-directed synthesis reaction in which a firstpolymerase exchanges with a second polymerase without terminating thereaction.

FIG. 4 provides a schematic depiction of an alternate embodiment of thecompositions in which a polymerase is covalently bound to asurface-immobilized template localizing moiety.

FIG. 5 provides a schematic depiction of a polynucleotide-containingtemplate localizing moiety that is complementary to a region of asingle-stranded, circular template nucleic acid and that forms a singleloop over the template upon dissociation.

FIG. 6 provides a schematic depiction of a polynucleotide-containingtemplate localizing moiety that is complementary to a region of asingle-stranded, circular template nucleic acid and that forms multipleloops around the template upon dissociation.

FIG. 7 provides a schematic depiction of a polynucleotide-containingtemplate localizing moiety that is complementary to a region of atemplate nucleic acid that comprises regions of internalcomplementarity.

DETAILED DESCRIPTION

OVERVIEW

Analysis of small reaction volumes, e.g., single-analyte moleculereactions, is becoming increasingly important in high throughputapplications, e.g., in nucleic acid sequencing. However, decreases inthe activity of individual sequencing enzyme molecules over time, canhave a detrimental effect on the real time analysis of the activity ofsuch sequencing enzymes, e.g., in a single-molecule sequencing reaction.The present invention is generally directed to compositions, methods,systems and kits that can be beneficially used to localize a sequencingenzyme to a reaction region, e.g., a ZMW, without necessarilyimmobilizing the sequencing enzyme itself, within or proximal to thereaction region. For example, a template localizing moiety, e.g., thatis capable of interacting with a sequencing enzyme, can be immobilizedon a solid surface, e.g., on a surface, a well, or a single-moleculereaction region, and can be used to fix a nucleic acid template to thesurface (see FIG. 1). For example, in certain preferred embodiments, themethods, compositions, and systems described herein are used withsingle-molecule sequencing technologies, in particular those describedin U.S. Pat. No. 7,056,661; Eid, et al. (2009) Science 299:682-686; andKorlach, et al. (2008) Nucleosides, Nucleotides and Nucleic Acids27:1072-1083, all of which are incorporated herein by reference in theirentireties for all purposes.

As used herein, a “template localizing moiety” is a moiety comprising,e.g., a natural or synthetic polymer, such as a protein other than apolymerase, or any of the discrete materials described herein, that canassociate with and/or retain a template nucleic acid (e.g., comprisingDNA, RNA, or analogs or derivatives thereof) and fix it to, e.g., thesurface on which the moiety itself has been immobilized. In someembodiments, a template localizing moiety can form a complex with asequencing enzyme in a manner that permits the activity of thesequencing enzyme on the template. In some embodiments, a templatelocalizing moiety can improve the processivity of a sequencing enzyme,and such moieties can include, e.g., a wide variety of DNA replicationfactors and/or DNA repair factors, as discussed hereinbelow.

Although certain descriptions of the invention herein are primarilyfocused on template-dependent sequencing-by-synthesis methods thatmonitor incorporation of labeled nucleotide analogs into a nascentstrand, it will be clear to one of ordinary skill upon review of theinstant disclosure that the template localizing moieties can be used toimmobilize template nucleic acids in myriad analytical reactions,including but not limited to exonuclease sequencing, pyrosequencing,nanopore-based sequencing, ligase-mediated sequencing, binding assays,and amplification-based methods. Such methods of known in the art andare further described, e.g., in WO/1994/023066; U.S. Pat. Nos.5,516,633, 5,622,824, 5,750,341, 5,795,782, 5,969,119, 6,210,891,6,258,568, 6,306,597, and 7,485,425; U.S. Ser. No. 61/186,661, filedJun. 12, 2009; and U.S. Patent Publication Nos. 2007115205 and20090131642, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

As shown in FIG. 1, template localizing moiety 110 is immobilized withinsingle molecule reaction region 100. Moiety 110 can fix template nucleicacid 120 to single molecule reaction region 100 to produce composition130. In some embodiments of the compositions provided by the invention,the moiety topologically encircles the template, e.g., surrounds andencloses the template. For example, template localizing moiety 110topologically encircles template 120 such that template 120 passesthrough moiety 110 not unlike a thread passes through the eye of aneedle.

The template nucleic acid of the compositions, e.g., a DNA or an RNA,can be linear (see FIG. 1) or, in preferred embodiments, it can becircular, e.g., form a “closed loop” wherein each nucleotide iscovalently joined to the nucleotides preceding and following it (seeFIG. 2). As shown in FIG. 2, template localizing moiety 210topologically encircles circular template nucleic acid 220, fixing itwithin single molecule reaction region 200. Closed nucleic acid loopsthat are fixed within or proximal to a reaction region, e.g., a ZMW,through a surface-immobilized template localizing moiety will notdiffuse out of the reaction region as readily as linear templates. Thisorientation of a template nucleic acid is particularly useful forredundant sequencing applications in which a single template issubjected to a sequencing reaction multiple times to generate multiplereplicate nucleotide sequences that correspond (e.g., are identical orcomplementary) to the template nucleic acid. For example, arolling-circle sequencing-by-synthesis reaction can be performed inwhich a polymerase capable of strand displacement repeatedly processes acircular template to synthesize a long, concatemeric nascent strand. Thesynthesis of the nascent strand is monitored to generate a longnucleotide sequence “read” for the nascent strand that contains multiplecopies of a sequence complementary to the template strand, and this readis subjected to statistical analysis to determine the sequence of thetemplate strand. Such rolling-circle synthesis can be used in othersequencing technologies, as well, such as pyrosequencing methods.

Typically, single molecule sequencing-by-synthesis reactions take placein the presence of one or more fluorescently labeled nucleotides and/ornucleotide analogues. In general, the incorporation or release of thefluorescent label can be used to indicate the presence and compositionof a growing nucleic acid strand, e.g., providing evidence oftemplate-directed synthesis and/or the sequence of the nascent strandbeing synthesized, and by complementarity, the sequence of the templatenucleic acid. As shown in FIG. 3, template localizing moiety 310, whichhas been immobilized within single molecule reaction region 300, hasassociated with and topologically encircled nucleic acid template 301,fixing it within the reaction region. Polymerase 330 can diffuse intothe reaction region to initiate template-directed synthesis of a nascentstrand that is complementary to at least a portion of a strand oftemplate 301 to produce nascent strand 340. As used herein, a “nascentstrand” is a nucleic acid molecule that is synthesized by a polymeraseenzyme during the processing of a strand of a template nucleic acid.Although it is sometimes termed a “copy” of the template strand, thenascent strand actually comprises a sequence complementary to that ofthe strand of the template nucleic acid. Likewise, template-directedsynthesis of a template nucleic acid is sometimes referred to as“replication” of the template nucleic acid, although the nascent strandsynthesized is complementary rather than identical to the templatenucleic acid. As such, one of ordinary skill will recognize thatreference to “replication” of a template nucleic acid includes synthesisof a nascent strand complementary to the template strand.

Over time, a polymerase's activity and fidelity can decrease. Forexample, prolonged exposure of a polymerase, e.g., polymerase 330, tothe optical energy of the fluorescently labeled nucleotides ornucleotide analogues that are incorporated into a nascent and growingnucleic acid, e.g., nascent strand 340 can reduce the enzyme'sprocessivity, accuracy, and polymerase activity over time (seecomposition 350, which includes inactive polymerase 335). Otherenvironmental factors that can lead to polymerase inactivation include,e.g., oxidation, degradation, and the like. Inactive polymerase 335dissociates from the template 301 and can exchange with activepolymerase 345 without terminating the sequencing read, e.g., thepolymerase-mediated processing of template 301 can reinitiate uponassociation with a second polymerase, e.g., active polymerase 345, tothe immobilized template 301. Typically, nascent strand 340 remains insingle molecule reaction region 300 during such a polymerase exchange sothat active polymerase 345 can continue incorporating nucleotides intonascent strand 340, e.g., using 301 as a template. In certainembodiments, nascent strand 340 can be removed from template 301 priorto reinitiation of template-directed synthesis by active polymerase 345,e.g., by heat-denaturation, chemical treatment, high salt concentration,etc. Since nascent strand 340 is held in reaction region 300 only byassociation with template nucleic acid 301, disruption of thatassociation facilitates removal of nascent strand 340 from reactionregion 300.

Optionally, a template localizing moiety can also form a complex with asequencing enzyme, e.g., to bring the sequencing enzyme to a portion ofthe template that is at a reaction site and/or within an observation (ordetection) volume. For example, in certain embodiments of thecompositions (see FIG. 4), a polymerase, e.g., polymerase 400 can becovalently attached to the surface-immobilized template localizingmoiety, e.g., moiety 410, e.g., via the polymerase's C-terminal end,e.g., polymerase C-terminal end 420. Alternatively, an exonuclease canbe brought into proximity to a terminal portion of a template nucleicacid. However, in preferred embodiments of the compositions, asequencing enzyme associates with the moiety in a non-covalent manner.Optionally, a sequencing enzyme can bind the template tethering moietyvia a reversibly cleavable linker, e.g., a linker that can reform with anew sequencing enzyme. This permits the sequencing enzyme to exchangewith other sequencing enzymes present, e.g., in a sequencing reactionmix, without terminating the sequencing reaction. In yet furtherembodiments, a sequencing enzyme can be covalently or non-covalentlyattached to a linker bound to the surface, and in certain preferredembodiments such a linker is a cleavable linker that allows release of asequencing enzyme, e.g., to facilitate exchange with another sequencingenzyme in the reaction mixture. In certain embodiments in which amultisubunit sequencing enzyme is used, all or only one or a subset ofsubunits can be attached to the template localizing moiety and/or thesurface. For example, HIV reverse transcriptase is a heterodimer andonly one of the subunits need be attached to the template localizingmoiety and/or surface in order to maintain the enzyme at the reactionsite. A reversible attachment, e.g., a photocleavable linker, can beused to facilitate sequencing enzyme exchange during the course of thereaction.

The compositions of the invention rely on a surface-immobilized templatelocalizing moiety, rather than a surface-immobilized sequencing enzyme,to localize a sequencing reaction, e.g., template-directed synthesis orexonuclease degradation reaction, to a defined reaction region.Sequencing reactions that include the provided compositions, e.g.,compositions in which a first, e.g., less active or inactive, sequencingenzyme can be exchanged with a second, e.g., active, sequencing enzymeare not terminated when a sequencing enzyme's activity, processivity,and fidelity decreases, e.g., as a result of the exposure to opticalenergy of fluorescently labeled nucleotides and/or nucleotide analogs.As a result, the methods and systems of the invention, in which thecompositions described above can be used, can beneficially increasesequence throughput and improve the accuracy of sequence data. Moreover,the invention can advantageously lower fabrication and reagent costs(see FIG. 1). For example, an array of single molecule reaction volumesin which individual sequencing enzymes have been immobilized is nolonger useful after the sequencing enzymes have become inactive.However, an array of single molecule reaction regions in whichindividual template localizing moieties have been immobilized, e.g.,FIG. 1, array 140, can be used repeatedly and continuously.

Further, in embodiments in which the sequencing enzyme is not tetheredto the surface or the template localizing moiety, the sequencing enzymeactivity may be enhanced by virtue of the lack of a physical linkage tothe sequencing enzyme. For example, a polymerase enzyme that is free insolution is not hindered by being directly tethered to a surface ortemplate localizing moiety, which may interfere with conformationalchanges required for template-directed synthesis, e.g., due to torsionalstress, electrostatic interference, or steric hindrance caused by thelinking moiety, potentially causing a decrease in activity,processivity, or accuracy of the enzyme. Further, a polymerase that isfree in solution can be a more “natural” polymerase than a polymerasecomprising structural alterations required for binding to the surface.In addition, a potential source of experimental variation is eliminatedsince there can be no variation due to differences in sequencing enzymeimmobilization chemistry between different reaction sites on the same ordifferent surfaces.

FURTHER DETAILS REGARDING TEMPLATE LOCALIZING MOIETIES

The compositions of the invention rely on a surface-immobilized templatelocalizing moiety, rather than a surface-immobilized polymerase, tolocalize a sequencing reaction, e.g., template-directed synthesis orexonuclease sequencing reaction, to a defined reaction region. Thisconfiguration can beneficially increase read lengths and improve theaccuracy of the sequencing data produced by e.g., a single moleculesequencing reaction, as it permits the exchange of a first, e.g.,inactive, e.g., photodamaged, sequencing enzyme with a second, e.g.,active, e.g., non-photodamaged, sequencing enzyme present in, e.g., asequencing reaction mix, without terminating nucleic acid sequencing(e.g., a template-directed synthesis reaction can proceed anew when anactive polymerase replaces a polymerase whose activity has decreased asa result of prolonged exposure to the optical energy of fluorescentlylabeled nucleotides and/or nucleotide analogs in the sequencing reactionmix.) Advantageously, the compositions of the invention can decreasereagent use and lower the fabrication costs of, e.g., ZMW arrays used inhigh-throughput single-molecule sequencing systems.

In some aspects, a template localizing moiety can comprise, e.g., apolymer, and/or any discrete material that can be coupled/associated, atleast temporarily, to or with a nucleic acid, e.g., a DNA or an RNA.Such a polymer can comprise natural structural units (e.g., nucleotides,amino acids, sugars, etc.), or synthetic structural units (e.g.,styrene, ethylene, propylene, etc.), or modifications and/orcombinations thereof. For example, such a polymer can comprise one ormore polynucleotides, polypeptides, polysaccharides, polystyrene,polyethylene (e.g., polyethylene glycol, Spacer 18, etc.),polypropylene, polymer beads, silica beads, ceramic beads, glass beads,magnetic beads, metallic beads, and organic resin beads can be used tolocalize a template nucleic acid to a defined reaction region. Suchtemplate localizing moieties can have essentially any shape, e.g.,spherical, helical, spheroid, rod shaped, cone shaped, disk shaped,cubic, polyhedral or a combination thereof. In preferred embodiments,the template localizing moiety topologically encircles the templatenucleic acid. Optionally, the shape of a template localizing moiety canalso be used to orient the moiety in the relevant well, e.g., to ensurethat the immobilized nucleic acid is accessible to a sequencing enzymeand can be used as a template in, e.g., a sequencing reaction. Templatelocalizing moieties can optionally be coupled to any of a variety ofreagents that facilitate surface attachment of the nucleic acid, e.g., aDNA or an RNA.

In certain preferred embodiments, a template localizing moiety canfunction not only to localize the template to a reaction region, butalso to effectively trap the sequencing enzyme in the observation ordetection volume of the reaction region. Take, for example, a templatelocalizing moiety large enough to allow passage of a template, but toosmall to allow passage of a polymerase. Upon encountering the templatelocalizing moiety, A polymerase translocating on the template would bespatially constrained at the template localizing moiety due to theinability to “follow” the template through the template localizingmoiety. Therefore, continued translocation along the template wouldrequire the template be pulled through the template localizing moiety bythe polymerase enzyme. Such template localizing moieties can comprisevarious types of polymers, including but not limited to polynucleotides,polypeptides, polysaccharides, and other synthetic polymers. Specificexamples using such template localizing moieties comprisingpolynucleotides and combinations of natural and synthetic polymers areprovided below.

Template localizing moieties of the invention can essentially be anydiscrete material that can be immobilized, e.g., on a planar surface, ina well, or in a single molecule reaction region, e.g., a ZMW. Desirably,the material(s) that comprises a template localizing moiety permit themoiety to associate with a template in such a manner that maintains orincreases a sequencing enzyme's processivity, e.g., in degrading thetemplate or performing template-directed nascent strand synthesis.Examples of such materials can include polymer beads or particles (e.g.,polystyrene, polypropylene, latex, nylon and many others), silica orsilicon beads, ceramic beads, glass beads, magnetic beads, metallicbeads and organic compound beads. An enormous variety of particles thatcan be used to fix a template to or near a defined reaction region arecommercially available, e.g., those typically used for chromatography(see, e.g., Catalogs from Sigma-Aldrich (Saint Louis, Mo.), SupelcoAnalytical (Bellefonte, Pa.; sold, e.g., through Sigma-Aldrich), as wellas those commonly used for affinity purification (e.g., the variousmagnetic Dynabeads™, which commonly include coupled reagents) suppliede.g. by Invitrogen. For a discussion of matrix materials see also, e.g.,Hagel et al. (2007) Handbook of Process Chromatography, Second EditionDevelopment, Manufacturing, Validation and Economics, Academic Press;2nd edition ISBN-10: 0123740231; Miller (2004) Chromatography: Conceptsand Contrasts Wiley-Interscience; 2nd edition ISBN-10: 0471472077;Satinder Ahuja (2002) Chromatography and Separation Science (SST)(Separation Science and Technology Academic Press, ISBN-10: 0120449811;Weiss (1995) Ion Chromatography VCH Publishers Inc.; Baker (1995)Capillary Electrophoresis John Wiley and Sons; Marcel Dekker and Scott(1995) Techniques and Practices of Chromatography Marcel Dekker, Inc.

In preferred embodiments of the compositions described herein, atemplate localizing moiety comprises a polypeptide, preferably a proteinother than a polymerase used to synthesize a polynucleotidecomplementary to the template nucleic acid, that can be attached to,e.g., a planar surface, a well, or a single-molecule reaction region,e.g., a ZMW, in an orientation that preserves its nucleic acid-bindingactivity and, optionally, its sequencing enzyme binding activity,wherein the protein is configured to form a complex with a sequencingenzyme. Proteins that can optimally be used as template localizingmoieties in the methods, compositions, systems, and kits of theinvention include a wide variety of DNA replication factors, DNA repairfactors, and/or transcription factors e.g., a processive nuclease, asingle-strand binding protein (SSBP), a helicase, a DNA repair enzyme, apolymerase mutant, fragment, or subunit thereof that lacks nascentstrand synthesis activity but is able to translocate along a templatenucleic acid, a DNA processivity factor, e.g., a helicase, or a proteinthat non-specifically binds a double-stranded nucleic acid—essentiallyany protein or protein mutant that can associate with a template nucleicacid and not interfere with an ongoing sequencing reaction. For example,human oxoguanine DNA glycosylase 1 (hOgg1), which is a DNAglycosylase/apurinic (AP) lyase (see, e.g., Klungland, et al. (2007) DNARepair (Amst) 6(4): 481-8, which is incorporated herein by reference inits entirety for all purposes) or homologs thereof, including yeast Oggproteins (e.g., yOgg1 or yOgg2), E. coli Mut proteins (e.g., MutM (FPGprotein), and others known in the art. Further, multiple such proteinsmay be bound at a single reaction site to immobilize a single templatemolecule.

As described above, a template localizing moiety of the compositionspreferably fixes a template nucleic acid to, e.g., a single moleculereaction region by topologically encircling the template (see, e.g.,FIG. 2 and corresponding description). For example, DNA polymerasesliding clamp proteins can be beneficially included in the compositionsof the invention. Sliding clamps are a family of multimeric ring-shapedDNA polymerase processivity factors that play essential roles in DNAmetabolism (reviewed in, e.g., Barsky, et al. (2005) “DNA slidingclamps: just the right twist to load onto DNA.” Curr Biol 15: R989-92and Indiani, et al. (2006) “The replication clamp-loading machine atwork in the three domains of life.” Nat Rev Mol Cell Biol 7: 751-761).Sliding clamp proteins have been identified in Bacteria, e.g., the βclamp of E. coli DNA polymerase III; Archea, e.g., archeal PCNA; andEukarya, e.g., eukaryal PCNA; as well as in viruses and phages, e.g., T7gp45.

Though they share little amino acid sequence homology, sliding clampsfrom Bacteria, Archea, and Eukaryotes have similar three-dimensionalstructures (Kelman, et al. (1995) “Structural and functionalsimilarities of prokaryotic and eukaryotic DNA polymerase slidingclamps.” Nucl Acid Res 23: 3613-3620; Iwai, et al. (2000) “Phylogeneticanalysis of archaeal PCNA homologues.” Extremophiles 4: 357-364; andHingorani, et al. (2000) “A tale of toroids in DNA metabolism.” Nat RevMol Cell Biol 1: 22-30). Sliding clamps comprise 2-3 monomers to yield aring comprised of six domains. Each ring has similar dimensions and acentral cavity large enough to accommodate a duplex DNA molecule(Kelman, et al. (1995) “Structural and functional similarities ofprokaryotic and eukaryotic DNA polymerase sliding clamps.” Nucl Acid Res23: 3613-3620 and Hingorani, et al. (2000) “A tale of toroids in DNAmetabolism.” Nat Rev Mol Cell Biol 1: 22-30).

Sliding clamp proteins are typically assembled around double-strandedDNA by a clamp loading complex (reviewed in O'Donnell, et al. (2002)“Clamp loaders and sliding clamps.” Curr Opin Struct Biol 12: 217-224)in an ATP-dependent reaction. Following assembly, sliding clamps canslide bidirectionally along the duplex (Stukenberg, et al. (1991)“Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme.”J Biol Chem 266: 11328-11334). Clamp proteins bind DNA polymerase andact as mobile tethers that prevent the enzyme from dissociating from atemplate DNA strand. Because a rate limiting step in DNA replication isthe association of the polymerase with the DNA template, the presence ofa sliding clamp can be beneficially increases the number of; e.g.,fluorescently labeled nucleotides that the polymerase can add to thegrowing strand per association event during, e.g., a sequencingreaction, thus increasing read length.

Additional details regarding sliding clamp proteins, clamp loadingcomplexes, and the DNA polymerases with which they interact areelaborated in, e.g., Georgescu, et al. (2008) “Structure of a SlidingClamp on DNA.” Cell 132: 43-54; Seybert, et al. (2004) “Distinct rolesfor ATP binding and hydrolysis at individual subunits of an archaealclamp loader.” EMBO J 23: 1360-1371; Bruck, et al. (2001) “The ring-typepolymerase sliding clamp family.” Genome Biol 2: reviews 3001.1-reviews3001.3; Johnson, et al. (2005) “Cellular DNA replicases: components anddynamics at the replication fork.” Annu Rev Biochem 74: 283-315; andVivona, et al. (2003) “The diverse spectrum of sliding clamp interactingproteins.” FEBS Lett 546:167-72. An artificial processivity clamp thatcan be bound to surfaces has recently been described in, e.g., Williams,et al. (2008) “An artificial processivity clamp made with streptavidinfacilitates oriented attachment of polymerase-DNA complexes tosurfaces.” Nucl Acids Res doi: 10.1093/nar/gkn531.

Hexameric helicases are another class of template localizing moietiesthat can be beneficially included in the methods, compositions, kits,and systems of the invention to, e.g., fix a template nucleic acid to asurface. Helicases can also form a processive complex with a DNApolymerase during processing of the template in, e.g., a sequencingreaction. Hexameric helicases, e.g., E. coli DnaB and Rho, T4 gp41, andT7 gp4, are a class of NTP-dependent motor proteins that play a role DNAmetabolism. Hexameric helicases have a characteristic ring-shapedstructure, and these enzymes typically move along the phosphodiesterbackbone of the nucleic acid to which they are bound, using the energyproduced by nucleic acid-stimulated NTP hydrolysis to translocate alongthe nucleic acid while catalyzing the unidirectional, processiveseparation of two strands of a complementary nucleic acid duplex. Recentstructural studies have indicated that a single strand of a DNA duplexpasses through the hexamer channel (Enemark, et al. (2006) “Mechanism ofDNA translocation in a replicative hexameric helicase,” Nature 442270-275).

A hexameric helicase can optimally be used with a non-processive,non-strand-displacing polymerase, e.g., a Klenow fragment, in, e.g., asequencing reaction. In certain embodiments that include a hexamerichelicase, the concentration of NTP present in. e.g., a sequencingreaction mix, can modulate the rate at which the helicase catalyzes theunwinding of a double-stranded DNA template. This, in turn, can modulatethe sequencing rate of, e.g., a non-strand displacing polymerase in atemplate-directed synthesis reaction.

Further details regarding hexameric helicase translocation mechanisms;hexameric helicase base pair separation mechanisms; and/or assays tomeasure helicase translocation rate or processivity are elaborated in,e.g., Enemark, et al. (2008) “On helicases and other motor proteins.”Curr Opin Strict Biol 18: 243-57, Epub March 2008; Sclafani, et al.(2004) “Two heads are better than one: regulation of DNA replication byhexameric helicases.” Genes Dev 18: 2039-2045; Patel, et al. (2000)“Structure and function of hexameric helicases.” Annu Rev Biochem 69:651-697; and Xie (2006) “Model for helicase translocating alongsingle-stranded DNA and unwinding double-stranded DNA.” Biochim BiophysActa 1764:1719-29, Epub 2006 Sep. 26.

In preferred embodiments of the compositions described herein, atemplate localizing moiety comprises a polynucleotide, i.e., apolynucleotide other than the template, that can be attached to, e.g., aplanar surface, a well, or a single-molecule reaction region, e.g., aZMW, in an orientation that allows it to constrain a template to whichit is initially annealed even after it has been displaced from thetemplate, e.g., by a translocating polymerase enzyme on the template.Polynucleotides that can optimally be used as template localizingmoieties in the methods comprise a central region that is complementaryto at least one region of the template to be immobilized and two endregions that associate with a surface of a reaction region such thatwhen bound to the surface the template localizing moiety loops over andoptionally completely around the template, thereby localizing it to thereaction site. The template can move through the loop(s) formed by thetemplate localizing moiety, but cannot diffuse away from the reactionregion unless either an end of the template localizing moiety isdissociated from the reaction region or an end of the template passesthrough the loop. As such, although linear templates can be used withsuch polynucleotide template localizing moieties, in certain embodimentsa circular template is preferred since a circular template can berepeatedly processed at a reaction region without “slipping out” of thetemplate localizing moiety. Further, if a polymerase dissociates fromthe template nucleic acid, a second polymerase can bind the template andcontinue template-directed synthesis using the same template nucleicacid at the same reaction region. Since the polymerase is not covalentlytethered, it can readily dissociate and exchange with another polymerasein the reaction mixture. As such, a damaged polymerase can be replacedby an undamaged polymerase, thereby allowing stalled synthesis tocontinue on the same template nucleic acid. Data generated bytemplate-directed synthesis using a single template nucleic acid bymultiple polymerases can thereby be generated and collectedsequentially, and subjected to statistical analysis to determine asequence of the template nucleic acid.

A strand of double-stranded DNA usually circles the axis of the doublehelix once every 10.4 base pairs. As such, in certain aspects, atemplate localizing moiety comprises a polynucleotide portion that iscomplementary to at least about ten or more adjacent nucleotides toensure that the complementary region wraps around the template strand atleast one time. In certain embodiments, the complementary region islonger to create multiple loops around the template strand. Further, incertain preferred embodiments, one or more loops formed by a templatelocalizing moiety around a template nucleic acid block passage of apolymerase enzyme translocating on the template, effectively localizingthe polymerase to the template at the template localizing moiety. Thiscan serve to position the polymerase at a desired location within areaction region, e.g., in the observation volume. This aspect isespecially useful for large template nucleic acids that extend outsidethe observation volume.

A further advantage provided by a template localizing moiety comprisinga portion complementary to a template nucleic acid is the ability toselectively immobilized a subset of template nucleic acids having one ormore particular polynucleotide sequences of interest (e.g., exonic orintronic regions, regulatory regions, and the like). For example, awhole genomic sample can be fragmented and mixed with a pool of templatelocalizing moeties having polynucleotide regions complementary to a setof genetic loci known to predict susceptibility to a given disease. Onlygenomic fragments having one or more of those genetic loci of interestwill be targeted and immobilized by the template localizing moieties,and subsequently subjected to sequence analysis. This strategysignificantly reduces the amount of data generated, and therefore theamount of statistical analysis required for determining the relevantgenotypes for an individual, and by association, their susceptibility tothe given disease.

FIG. 5 provides an exemplary embodiment of a polynucleotide-containingtemplate localizing moiety 510 that comprises a polynucleotide regioncomplementary to a region of a single-stranded, circular templatenucleic acid 520 long enough to loop over the template nucleic acid 520one time. The ends of the template localizing moiety 510 are derivatizedwith biotin 560 to promote binding of the ends of the templatelocalizing moiety 510 to the streptavidin tetramer 550. The templatelocalizing moiety 510 is annealed to the template nucleic acid 520, andis subsequently immobilized on a substrate 540 via interaction with astreptavidin tetramer 550 bound to a biotin-derivatized surface of thesubstrate 540. The template nucleic acid 520 is also annealed to primer570, and subsequently exposed to a polymerase 530. Binding of polymerase530 to the complex results in extension of the primer 570 as thepolymerase translocates along the template nucleic acid 520, producing anascent polynucleotide strand 580. Upon displacement of thecomplementary region of the template localizing moiety 510, a singleloop is formed that passes over the template nucleic acid 520, therebylocalizing it to the reaction region on the substrate 540. Arrow 590shows the direction of movement of the template strand 520 toward thepolymerase 530 during translocation when the polymerase 530 is blockedby the template localizing moiety 510. Although FIG. 5 illustrates anembodiment in which a single subunit of the streptavidin tetramer 550 islinked to the surface and two are linked to the template localizingmoiety 510, further embodiments include utilization of the fourthsubunit, e.g., to link to the surface, the sequencing enzyme, or othercomponents of a reaction mixture, including but not limited toelongation factors, components of a detection system (e.g.,luciferase/sulfurylase), etc.

FIG. 6 provides an exemplary embodiment of a polynucleotide-containingtemplate localizing moiety 610 that comprises a polynucleotide regioncomplementary to a region of a single-stranded, circular templatenucleic acid 620 long enough to loop over the template nucleic acid 620three times. The ends of the template localizing moiety 610 arederivatized with biotin 660 to promote binding of the ends of thetemplate localizing moiety 610 to the streptavidin tetramer 650. Thetemplate localizing moiety 610 is annealed to the template nucleic acid620, and is subsequently immobilized on a substrate 640 via interactionwith a streptavidin tetramer 650 bound to a biotin-derivatized surfaceof the substrate 640. The template nucleic acid 620 is also annealed toprimer 670, and subsequently exposed to a polymerase 630. Binding ofpolymerase 630 to the complex results in extension of the primer 670 asthe polymerase translocates along the template nucleic acid 620,producing a nascent polynucleotide strand 680. Upon displacement of thecomplementary region of the template localizing moiety 610, a singleloop is formed that passes over the template nucleic acid 620, therebylocalizing it to the reaction region on the substrate 640. Arrow 690shows the direction of movement of the template strand 620 toward thepolymerase 630 during translocation when the polymerase 630 is blockedby the template localizing moiety 610.

FIG. 7 provides an exemplary embodiment of a polynucleotide-containingtemplate localizing moiety 710 that comprises a polynucleotide regioncomplementary to a region of a single-stranded, circular templatenucleic acid 720 long enough to loop over the template nucleic acid 720three times. However, unlike the embodiment depicted in FIG. 6, thetemplate nucleic acid 720 comprises regions of internal complementarity(shown as double-stranded region 725), such that it can form a partiallydouble-stranded template nucleic acid. The ends of the templatelocalizing moiety 710 are derivatized with biotin 760 to promote bindingof the ends of the template localizing moiety 710 to the streptavidintetramer 750. Primer 770 and template localizing moiety 710 are annealedto template nucleic acid 720, e.g., following heat-denaturation. In somepreferred embodiments, template localizing moiety 710 is annealed to onestrand within the duplex region of the template nucleic acid 720. Theresulting annealed complex is subsequently immobilized on a substrate740 via interaction with the streptavidin tetramer 750 bound to abiotin-derivatized surface of the substrate 740. The template nucleicacid 720 is subsequently exposed to a polymerase 730, which extendsprimer 770 as the polymerase translocates along the template nucleicacid 720, separating any duplex regions in its path and producing anascent polynucleotide strand 780. Upon displacement of thecomplementary region of the template localizing moiety 710, three loopsare formed that pass over the template nucleic acid 720, therebylocalizing it to the reaction region on the substrate 740. Arrow 790shows the direction of movement of the template strand 720 toward thepolymerase 730 during translocation when the polymerase 730 is blockedby the template localizing moiety 710 looped around the template nucleicacid.

In some embodiments, the template nucleic acid 720 comprises a tagsequence 795 in the single-stranded region that can be used to identifycertain characteristics of the template nucleic acid 720, e.g., sourceinformation. For example, a genomic DNA sample can be fragmented toproduce a set of double-stranded DNA fragments, and each fragment can belinked to two single-stranded hairpins, one at each end. A tag sequenceincorporated into at least one of the hairpin structures contains anucleotide sequence that identifies the source (e.g., individual,species, subspecies, experimental/clinical group, etc.) from which thegenomic DNA was isolated. Such tag sequences allow pooling of samplesfrom various sources where the sample from each source is differentiallytagged. During sequence analysis, the identification of a particular tagsequence in the sequencing read is used to deconvolute the pooledsequencing data and identify the particular source of the sample. Suchtag sequences (also termed “registration sequences”) and partiallydouble-stranded template nucleic acids are further described in U.S.patent application Ser. No. 12/413,258, filed Mar. 27, 2009, which isincorporated herein by reference in its entirety for all purposes.

Although described above primarily in terms of biotin-streptavidinlinkages, a polynucleotide template localizing moiety can be derivatizedat each end with other entities that preferentially associate with amolecule immobilized at a reaction region. For example, each end of atemplate localizing moiety can be derivatized with a chemically activelinkage including but not limited to “Click Chemistry” (Kolb, et al.(2001) Angew. Chem. Int. Ed. 40:2004-2021; and CLIP- and SNAP-tagstrategies (New England BioLabs, Inc.). Further, a variety of surfaceattachment strategies can be used, including disulfide bond formation,amine linkages through an activated carbonyl, reactive groups on anumber of siloxane functionalizing reagents (described elsewhereherein), and the like.

In certain preferred embodiments, a template localizing moiety thatcomprises a polynucleotide portion that is complementary to a templatenucleic acid also comprises one or more polynucleotide portions that arenot complementary to the template nucleic acid and/or one or moreportions that do not comprise polynucleotides. In certain embodiments,one or more ends of the complementary portion may be linked tonon-complementary portions, e.g., poly-T, poly-A, and the like. In otherembodiments, a complementary polynucleotide portion may be flanked byportions comprising synthetic structural units, e.g., polyethyleneglycol, Spacer 18 (Integrated DNA Technologies), and the like. Spacer 18is an 18-atom hexa-ethyleneglycol spacer (shown below) and, in certainembodiments, between two and five units of Spacer 18 is linked to eachend of the polynucleotide portion of a template localizing moiety.

In yet further embodiments, a template localizing moiety comprises bothone or more non-complementary polynucleotide portions and one or moresynthetic polymer portions. Benefits from such hybrid structures aremyriad and include less costly synthesis of the synthetic structuralunits and reduced potential for interference with a translocatingpolymerase. Further, the shape and/or stiffness of the portion of thetemplate localizing moiety that bind, directly or indirectly, to thereaction region can be modified based upon the natural and/or syntheticstructural unit composition. The biochemical characteristics of suchstructural units, as well as the chemical synthesis methods to linkthem, are well understood to those of ordinary skill in the art.FURTHER DETAILS REGARDING COUPLING TEMPLATE LOCALIZING MOIETIES TOSURFACES

The compositions of the invention include a template localizing moietythat has been immobilized, e.g., on a planar surface, in a well, or in asingle-molecular reaction region, e.g., a zero-mode waveguide (ZMW). Inembodiments where the moiety comprises a protein, the protein ispreferably immobilized in an orientation that preserves the protein'sability to bind/associate with a nucleic acid and, and in someembodiments form a complex with a sequencing enzyme. The immobilizedtemplate localizing moiety can fix a template nucleic acid to thesurface and can thereby advantageously localize, e.g., a DNA sequencingreaction, e.g., a template-directed synthesis reaction, to a definedreaction site. As described elsewhere herein, such compositions canbeneficially increase the lengths and accuracy of sequencing reads andlower fabrication costs and reagent use when used in, e.g.,high-throughput single-molecule sequencing systems.

In some embodiments, the template localizing moiety can interactdirectly with a surface, as described below. Alternatively or inaddition, a wide variety of linking chemistries are available forlinking template localizing moieties, e.g., those described herein, to awide variety of molecular, solid or semi-solid support elements. Thesechemistries can be performed in situ (i.e., in the reaction region inwhich the protein is to be immobilized) or prior to introduction of thetemplate localizing moiety into the well or reaction region. It isimpractical and unnecessary to describe all of the possible knownlinking chemistries for linking proteins to a solid support. It isexpected that one of skill can easily select appropriate chemistries,depending on the intended application.

In one preferred embodiment, the surfaces to which a template localizingmoiety is coupled comprise silicate elements (e.g., an array of ZMWsfabricated from glass or silicate compounds). A variety of silicon-basedmolecules appropriate for functionalizing surfaces are commerciallyavailable. See, for example, Silicon Compounds Registry and Review,United Chemical Technologies, Bristol, Pa. Additionally, the art in thisarea is very well developed and those of skill will be able to choose anappropriate molecule for a given purpose. Appropriate molecules can bepurchased commercially, synthesized de novo, or it can be formed bymodifying an available molecule to produce one having the desiredstructure and/or characteristics.

The substrate linker attaches to the solid substrate through any of avariety of chemical bonds. For example, the linker is optionallyattached to the solid substrate using carbon-carbon bonds, for examplevia substrates having (poly)trifluorochloroethylene surfaces, orsiloxane bonds (using, for example, glass or silicon oxide as the solidsubstrate). Siloxane bonds with the surface of the substrate are formedin one embodiment via reactions of derivatization reagents bearingtrichlorosilyl or trialkoxysilyl groups. The particular linking group isselected based upon, e.g., its hydrophilic/hydrophobic properties wherepresentation of an attached polymer in solution is desirable. Groupswhich are suitable for attachment to a linking group include amine,hydroxyl, thiol (e.g., in the case of gold surfaces), carboxylic acid,ester, amide, isocyanate and isothiocyanate. Preferred derivatizinggroups include aminoalkyltrialkoxysilanes, hydroxyalkyltrialkoxysilanes,polyethyleneglycols, polyethylene imine, polyacrylamide,polyvinylalcohol and combinations thereof.

By way of non-limiting example, the reactive groups on a number ofsiloxane functionalizing reagents can be converted to other usefulfunctional groups:

-   -   1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with        diborane, and H2O2 to oxidize the alcohol);        -   a. allyl trichlorosilane→→3-hydroxypropyl        -   b. 7-oct-1-enyl trichlorchlorosilane→→8-hydroxyoctyl    -   2. Diol (dihydroxyalkyl) siloxanes (silylate surface and        hydrolyze to diol)        -   a. (glycidyl            trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl    -   3. Aminoalkyl siloxanes (amines requiring no intermediate        functionalizing step)        -   a. 3-aminopropyl trimethoxysilane→aminopropyl    -   4. Dimeric secondary aminoalkyl siloxanes        -   a. bis(3-trimethoxysilylpropyl)→amine            bis(silyloxylpropyl)amine.

See, for example, Leyden et al., Symposium on Silylated Surfaces, Gordon& Breach 1980; Arkles, Chemtech 7, 766 (1977); and Plueddemann, SilaneCoupling Reagents, Plenum, N.Y., 1982. These examples are illustrativeand do not limit the types of reactive group interconversions which areuseful in conjunction with the present invention. Additional startingmaterials and reaction schemes will be apparent to those of skill in theart.

Template localizing moieties bearing a surface-exposed charge can thenbe coupled to a derivatized surface, e.g., planar surface, well, orsingle-molecule reaction region, e.g., ZMW. For example, the chargedgroup can be a carboxylate, quaternary amine or protonated amine that isa component of e.g., an amino acid that has a charged or potentiallycharged side chain. The amino acids can be either those having astructure which occurs naturally or they can be of unnatural structure(i.e., synthetic). Useful naturally occurring amino acids include:arginine, lysine, aspartic acid and glutamic acid. Surfaces utilizing acombination of these amino acids can be of use in the present invention.Further, peptides comprising one or more residues having a charged orpotentially charged side chain are useful coating components and theycan be synthesized utilizing arginine, lysine, aspartic acid, glutamicacid and combinations thereof. Useful unnatural amino acids arecommercially available or can be synthesized utilizing art-recognizedmethodologies, such as available systems of orthogonal elements. Inthose embodiments in which an amino acid moiety having an acidic orbasic side chain is used, these moieties can be attached to a surfacebearing a reactive group through standard peptide synthesismethodologies or easily accessible variations thereof. See, for example,Jones, Amino Acid and Peptide Synthesis, Oxford University Press,Oxford, 1992.

Linking groups can also be placed on surfaces to which a templatelocalizing moiety is to be immobilized. Linking groups of use in thepresent invention can have a range of structures, substituents andsubstitution patterns. They can, for example be derivatized withnitrogen, oxygen and/or sulfur containing groups which are pendent from,or integral to, the linker group backbone. Examples include, polyethers,polyacids (polyacrylic acid, polylactic acid), polyols (e.g.,glycerol,), polyamines (e.g., spermine, spermidine) and molecules havingmore than one nitrogen, oxygen and/or sulfur moiety (e.g.,1,3-diamino-2-propanol, taurine).

In some aspects, the coupling chemistries for coupling a templatelocalizing moiety to a surface of interest can be light-controllable,i.e., utilize photo-reactive chemistries. The use of photo-reactivechemistries and masking strategies to activate coupling of molecules,e.g., template localizing moieties, to substrates, as well as otherphoto-reactive chemistries is generally known (e.g., for semi-conductorchip fabrication and for coupling bio-polymers to solid phasematerials). Among a wide variety of protecting groups which are usefulare nitroveratryl (NVOC)-methylnitroveratryl (Menvoc), allyloxycarbonyl(ALLOC), fluorenylmethoxycarbonyl (FMOC),-methylnitro-piperonyloxycarbonyl (MeNPOC), —NH-FMOC groups, t-butylesters, t-butyl ethers, and the like. Various exemplary protectinggroups (including both photo-cleavable and non-photo-cleavable groups)are described in, for example, Atherton et al., (1989) Solid PhasePeptide Synthesis, IRL Press, and Greene, et al. (1991) ProtectiveGroups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y.The use of these and other photo-cleavable linking groups for nucleicacid and peptide synthesis on solid supports is a well-establishedmethodology.

Devices, methods and systems that incorporate functionalized regionsinto the walls of a ZMW, e.g., by incorporating an annular gold ringinto the walls of the ZMW, are described, e.g., in Foquet et al.SUBSTRATES AND METHODS FOR SELECTIVE IMMOBILIZATION OF ACTIVE MOLECULES(U.S. Ser. No. 60/905,786, filed Mar. 7, 2007 and U.S. PatentPublication No. 20080220537), incorporated herein by reference in theirentireties for all purposes.

Template localizing moieties can include appropriate functionalities forlinking to the relevant array surface. For example, thiol chemistriescan be used to link, e.g., a template localizing moiety to, e.g., aplanar surface, a well, or a single molecule reaction region. Templatelocalizing moieties can include linking groups, e.g., one or more biotintags, SNAP tags, CLIP tags, or a combination thereof, all of which areknown in the art and commercially available. For example, a templatelocalizing moiety can comprise a fusion protein between a sliding clampprotein and a biotin tag that facilitates immobilization of the slidingclamp protein by binding to streptavidin on the surface. Templatelocalizing moieties that comprise recombinantly expressed proteins canalso include unnatural amino acids with any of a variety of linkingchemistries, e.g., when expressed in a host cell that includesorthogonal elements that permit site-specific expression of theunnatural amino acid. Systems of orthogonal elements that can be used toincorporate unnatural amino acids, including amino acids with reactivegroups, are described in Wang, et al. (2006) “Expanding the geneticcode.” Annu Rev Biophys Biomolec Struct 35: 225-249; Wang and Schultz(2005) “Expanding the Genetic Code,” Angewandte Chemie Int. Ed.44(1):34-66; Xie, et al. (2005) “An expanding genetic code.” Methods 36:227-38; and Xie, et al. (2006) “A chemical toolkit for proteins: anexpanded genetic code.” Nat Rev Mol Cell Biol 7: 775-82.

The site-specific incorporation of an amino acid that comprises areactive/linking group can be used to specifically orient, e.g., atemplate localizing moiety that comprises a protein, relative to a wellor single molecule reaction region. Most preferably, such a protein isimmobilized in, e.g., a ZMW, in an orientation that permits the proteinto retain its activity, e.g., its ability to bind/associate with atemplate nucleic acid and, e.g., form a complex with a polymerase. Forexample, the well or reaction region can include a specificfunctionalized region (e.g., a gold band, as discussed above) that canbe coupled to a specific portion of the template localizing moiety.Additional useful strategies for coupling proteins to surfaces aredetailed in, e.g., WO 2007/076057 PROTEIN ENGINEERING STRATEGIES TOOPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al.

SEQUENCING ENZYMES

The invention provides compositions that include a localizing moiety on,e.g., a planar surface, a well, or a single molecule reaction region.Such compositions can be useful in fixing a template nucleic acid to thesurface, e.g., by topologically encircling the template, and localizingthe template to, e.g., a defined reaction region, e.g., asingle-molecule reaction volume. A template localizing moiety cancomprise a polymer, e.g., a protein other than a polymerase, and inparticular other than a polymerase used as a sequencing enzyme, e.g., toperform template-directed sequencing-by-synthesis. In certainembodiments of the invention, a sequencing enzyme can be engineered tocovalently bind to a template localizing moiety, e.g., via apolymerase's C-terminal end (see FIG. 4 and corresponding description).Optionally, a sequencing enzyme can be temporarily tethered to atemplate localizing moiety via, e.g., a reversibly cleavable linker,e.g., a linker that can reform with a new sequencing enzyme. In certainpreferred embodiments, the template localizing moiety is configured tonon-covalently associate with a sequencing enzyme, or to associateexclusively with the template and not with the sequencing enzyme. Incertain embodiments, a sequencing enzyme included in the compositionscan process a portion of at least one strand of the fixed template andexchange with a second sequencing enzyme, e.g., without terminating thesequencing reaction. The exchange of sequencing enzymes during nucleicacid sequencing reactions can be particularly beneficial in, e.g.,single-molecule template-directed synthesis reactions, e.g., performedin a ZMW, where a polymerase's processivity, accuracy, and polymeraseactivity can decrease over time. In one example, a DNA polymerase thathas sustained photodamage can exchange with a non-photodamaged DNApolymerase without disrupting the sequencing read (see FIG. 3 andcorresponding description), thus maintaining the accuracy with which thecorrect nucleotide is incorporated into a newly synthesized nucleic acidand/or increasing sequence throughput.

The exchange of polymerases is also beneficial where different types ofpolymerases are present in a reaction mixture, e.g., as in the JumpStartRED HT RT-PCR kit (Sigma-Aldrich®). In certain embodiments, more thanone polymerase may be present in a template-directed sequencing reactionin which one or more lesions may be present on the template nucleicacid. For example, “bypass polymerases” have been discovered in bothprokaryotes and eukaryotes, most of which belong to the Y-family ofpolymerases and/or are considered to be repair polymerases. In contrastto replicative polymerases, they operate at low speed, low fidelity, andlow processivity. However, because their active sites adopt a more openconfiguration than replicative polymerases they are less stringent andcan accommodate altered bases in their active sites. For moreinformation on bypass polymerases, see, e.g., Cordonnier, et al. (1999)Mol Cell Biol 19(3):2206-11; Friedberg, et al. (2005) Nat Rev Mal CellBiol 6(12):943-53; Holmquist, et al. (2002) Mutat Res 510(1-2):1-7;Lehmann, A. R. (2002) Mutat Res 509(1-2):23-34; Lehmann, A. R. (2006)Exp Cell Res 312(14):2673-6; Masutani, et al. (1999) Nature399(6737):700-4; and Ohmori, et al. (2001) Mol Cell 8(1):7-8, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes. Certain of these polymerases can bypasslesions in a nucleic acid template and carry out “translesion synthesis”or TLS. As such, DNA replication in the presence of such lesions wasfound to require multiple polymerases and the “polymerase switch model”was developed (see, e.g., Friedberg, et al. (2005) Nat Rev Mol Cell Biol6(12):943-53; Kannouche, et al. (2004) Cell Cycle 3(8):1011-3;Kannouche, et al. (2004) Mol Cell 14(4):491-500; and Lehmann, et al.(2007) DNA Repair (Amst) 6(7):891-9, all of which are incorporatedherein by reference in their entireties for all purposes). In brief, thepolymerase switch model is model for lesion bypass during replicationthat involves replacement of a replicative polymerase with a bypasspolymerase at a lesion, synthesis of the nascent strand by the bypasspolymerase until past the lesion, and subsequent replacement of thebypass polymerase with the more processive, higher fidelity replicativepolymerase for continued replication past the lesion. For example,during the course of a reaction in which a replicative polymeraseencounters and is blocked by a lesion in a template nucleic acid, thereplicative polymerase is replaced by a bypass polymerase at the site ofthe lesion, and the bypass polymerase synthesizes a segment of thenascent strand that is capable of base-pairing with the damaged base,and may further include one or more bases prior to and/or past the siteof the lesion in a process called “translesion synthesis.” The limitedprocessivity of the bypass polymerase causes it to dissociate and bereplaced by the replicative polymerase following translesion synthesis.The replicative polymerase continues to synthesize the nascent stranduntil another blocking lesion is encountered in the template, at whichpoint it is once again replaced by a bypass polymerase for translesionsynthesis. (See, e.g., Friedberg, et al. (2005) Nat Rev Mol Cell Biol6(12):943-53; and Kannouche, et al. (2004) Mol Cell 14(4):491-500,incorporated herein by reference above.) The process continues until thetemplate has been fully processed or the reaction is terminated, e.g.,by the investigator. One particular advantage of the polymerase switchmethod of template-dependent sequencing is that is it tolerant of mosttypes of lesions in the template nucleic acid. As such the damagedtemplate can be sequenced through a lesion, thereby allowingreinitiation of synthesis downstream of the lesion and increasing readlengths on lesion-containing templates.

Various different bypass polymerases known to those of ordinary skill inthe art can be used with the methods and compositions provided herein,include prokaryotic polymerases (e.g., DNA polymerase IV, polymerase V,Dpo4, Dbh, and UmuC) and eukaryotic polymerases (e.g., DNA polymerase η,DNA polymerase ι, DNA polymerase κ, and Rev1). In eukaryotes, multiplebypass polymerases participate in translesion synthesis, and aprocessivity factor, proliferating cell nuclear antigen (“PCNA”), isalso required and can be included in a sequencing reaction.

Monitoring reactions in which a template comprises damage or otherlesions generates data that can be statistically analyzed to determinethe number and locations of lesions in the template, and can potentiallyidentify the type of lesion. Since the portion of the nascent strandcorresponding to the site of the lesion in the template is synthesizedby a bypass polymerase, the sequence reads generated therefrom areexpected to be less reliable than those generated from regions of thenascent strand synthesized by the replicative polymerase. As such,redundancy in the sequencing reaction is may be a preferred means ofgenerating complete and accurate sequence reads. Redundancy can beachieved in various ways, including carrying out multiple sequencingreactions using the same original template, e.g., in an array format,e.g., a ZMW array. In some embodiments in which a lesion is unlikely tooccur in all the copies of a given template, the sequence data generatedin the multiple reactions can be combined and subjected to statisticalanalysis to determine a consensus sequence for the template. In thisway, the sequence data generated by processing the template with a lowerfidelity bypass polymerase can be supplemented and/or corrected withsequence data generated by processing the same template with a higherfidelity replicative polymerase. Alternatively or additionally, atemplate can be subjected to repeated sequencing reactions to generateredundant sequence information that can be analyzed to more thoroughlycharacterize the lesion(s) present in the template, e.g., by using asingle-stranded circular template nucleic acid immobilized at a reactionsite by various methods described elsewhere herein. Methods for templatedamage detection and bypass are further described in U.S. Ser. No.61/186,661, filed Jun. 12, 2009, and incorporated herein by reference inits entirety for all purposes.

DNA polymerases are sometimes classified into six main groups based uponvarious phylogenetic relationships, e.g., with E. coli Pol I (class A),E. coli Pol II (class B), E. coli Pol III (class C), Euryarchaeotic PolII (class D), human Pol beta (class X), and E. coli UmuC/DinB andeukaryotic RAD30/xeroderma pigmentosum variant (class Y). For a reviewof recent nomenclature, see, e.g., Burgers et al. (2001) “Eukaryotic DNApolymerases: proposal for a revised nomenclature” J Biol Chem 276:43487-90. For a review of polymerases, see, e.g., Hübscher et al. (2002)“Eukaryotic DNA Polymerases” Annual Review of Biochemistry Vol. 71:133-163; Alba (2001) “Protein Family Review: Replicative DNAPolymerases” Genome Biology 2(1): reviews 3002.1-3002.4; and Steitz(1999) “DNA polymerases: structural diversity and common mechanisms” JBiol Chem 274:17395-17398. The basic mechanisms of action for manypolymerases have been determined. The sequences of literally hundreds ofpolymerases are publicly available, and the crystal structures for manyof these have been determined, or can be inferred based upon similarityto solved crystal structures for homologous polymerases. For example,the crystal structure of Φ29, a preferred type of parental enzyme to bemodified according to the invention, is available. (See, e.g., Berman etal. (2007) EMBO J 26:3494-3505, Kamtekar et al. (2006) EMBO J25:1335-1343, and Kamtekar et al. (2004) Mol Cell 16:609-618.)

Structure/function analysis has revealed that most DNA polymerasescomprise a separate exonuclease domain. Many DNA polymerase enzymes havebeen modified in any of a variety of ways, e.g., to reduce or eliminateexonuclease activities (many native DNA polymerases have a proof-readingexonuclease function that interferes with, e.g., sequencingapplications), to simplify production by making protease digested enzymefragments such as the Klenow fragment recombinant, etc. DNA polymeraseshave also been modified to confer improvements in specificity,processivity, and improved retention time of labeled nucleotides inpolymerase-DNA-nucleotide complexes (e.g., WO 2007/076057 POLYMERASESFOR NUCLEOTIDE ANALOG INCORPORATION by Hanzel et al., andPCT/US2007/022459 POLYMERASE ENZYMES AND REAGENTS FOR ENHANCED NUCLEICACID SEQUENCING by Rank et al.), to improve surface-immobilized enzymeactivities (e.g., WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES byHanzel et al., and WO 2007/076057 PROTEIN ENGINEERING STRATEGIES TOOPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al.), toincrease closed complex stability and/or reduce branching rate (e.g.,61/072,645 GENERATION OF POLYMERASES WITH IMPROVED CLOSED COMPLEXSTABILITY AND DECREASED BRANCHING RATE by Clark, et al.), and to reducesusceptibility to photodamage (e.g., 61/072,643 ENZYMES RESISTANT TOPHOTODAMAGE by Bjornson, et al.). Any of these available polymerases canincluded with the surface-immobilized template localizing moiety in thecompositions, methods or systems of the invention to, e.g., improve theaccuracy of sequencing data and/or increase the read lengths ofsequencing reactions.

Many such polymerases are available, e.g., for use in sequencing,labeling and amplification technologies. For example, Human DNAPolymerase Beta is available from R&D systems. DNA polymerase I isavailable from Epicenter, GE Health Care, Invitrogen, New EnglandBiolabs, Promega, Roche Applied Science, Sigma Aldrich and many others.The Klenow fragment of DNA Polymerase I is available in both recombinantand protease digested versions, from, e.g., Ambion, Chimerx, eEnzymeLLC, GE Health Care, Invitrogen, New England Biolabs, Promega, RocheApplied Science, Sigma Aldrich and many others. Φ29 DNA polymerase isavailable from e.g., Epicentre. Poly A polymerase, reversetranscriptase, Sequenase, SP6 DNA polymerase, T4 DNA polymerase, T7 DNApolymerase, and a variety of thermostable DNA polymerases (Taq, hotstart, titanium Taq, etc.) are available from a variety of these andother sources. Recent commercial DNA polymerases include Phusion™High-Fidelity DNA Polymerase, available from New England Biolabs; GoTaq®Fiexi DNA Polymerase, available from Promega; RepliPHI™ Φ29 DNAPolymerase, available from Epicentre Biotechnologies; PfuUltra™ HotstartDNA Polymerase, available from Stratagene; KOD HiFi DNA Polymerase,available from Novagen; and many others. Biocompare(dot)com providescomparisons of many different commercially available polymerases.

DNA polymerases that are preferably included in the methods,compositions, and/or systems of the invention, e.g., to increase theread lengths of sequencing reactions, include Taq polymerases,exonuclease deficient Taq polymerases, E. coli DNA Polymerase 1, Klenowfragment, reverse transcriptases, Φ29 related polymerases including wildtype Φ29 polymerase and derivatives of such polymerases such asexonuclease deficient forms, T7 DNA polymerase, T5 DNA polymerase, anRB69 polymerase, etc. Further, in certain preferred embodiments,polymerases that are preferably included in the methods, compositions,and/or systems of the invention are capable of strand displacement. Avariety of strand displacing polymerase enzymes are readily available,including, for example, Φ29 polymerase and Φ29-type polymerases (See,e.g., U.S. Pat. Nos. 5,001,050, 5,576,204, the full disclosures of whichare incorporated herein by reference in their entirety for allpurposes), Bst polymerase (available from New England Biolabs), as wellas those polymerases described in commonly owned International PatentApplication Nos. WO 2007/075987, WO 2007/075873, WO 2007/076057 the fulldisclosures of which are incorporated herein by reference in theirentirety for all purposes.

In one aspect, the polymerase that is included with an immobilizedtemplate localizing moiety in the methods, compositions and/or systemsof the invention is a Φ29-type DNA polymerase. For example, the modifiedrecombinant DNA polymerase can be homologous to a wild-type orexonuclease deficient Φ29 DNA polymerase, e.g., as described in U.S.Pat. No. 5,001,050, 5,198,543, or 5,576,204. Alternately, DNA polymeraseof the methods, systems, and/or compositions can be homologous to otherΦ29-type DNA polymerases, such as B103, GA-1, PZA, Φ15, BS32, M2Y, Nf,G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, Φ21, or thelike. For nomenclature, see also, Meijer et al. (2001) “Φ29 Family ofPhages” Microbiology and Molecular Biology Reviews, 65(2): 261-287.

In addition to wild-type polymerases, chimeric polymerases made from amosaic of different sources can be included in the compositions andmethods described herein. For example, Φ29 polymerases made takingsequences from more than one parental polymerase into account can beused as a starting point for mutation to produce the polymerases of theinvention. This can done, e.g., using consideration of similarityregions between the polymerases to define consensus sequences that areused in the chimera, or using gene shuffling technologies in whichmultiple Φ29-related polymerases are randomly or semi-randomly shuffledvia available gene shuffling techniques (e.g., via “family geneshuffling”; see Crameri et al. (1998) “DNA shuffling of a family ofgenes from diverse species accelerates directed evolution” Nature391:288-291; Clackson et al. (1991) “Making antibody fragments usingphage display libraries” Nature 352:624-628; Gibbs et al. (2001)“Degenerate oligonucleotide gene shuffling (DOGS): a method forenhancing the frequency of recombination with family shuffling” Gene271:13-20; and Hiraga and Arnold (2003) “General method forsequence-independent site-directed chimeragenesis: J. Mol. Biol.330:287-296). In these methods, the recombination points can bepredetermined such that the gene fragments assemble in the correctorder. However, the combinations, e.g., chimeras, can be formed atrandom. Using the methods described above, a chimeric polymerase, e.g.,comprising segments of a B103 polymerase, a GA-1 polymerase, a PZApolymerase, a Φ15 polymerase, a BS32 polymerase, a M2Y polymerase, an Nfpolymerase, a G1 polymerase, a Cp-1 polymerase, a PRD1 polymerase, a PZEpolymerase, an SF5 polymerase, a Cp-5 polymerase, a Cp-7 polymerase, aPR4 polymerase, a PR5 polymerase, a PR722 polymerase, an L17 polymerase,and/or an F21 polymerase, can be generated for use with templatelocalizing moieties in compositions and methods provided by theinvention.

As described above, template localization moieties are also useful inexonuclease sequencing applications. Briefly, exonuclease sequencingdetermines the sequence of a nucleic acid by degrading the nucleic acidunilaterally from a first end with an exonuclease to sequentiallyrelease individual nucleotides. Each of the sequentially releasednucleotides is identified, e.g., by mass spectrometry, and the sequenceof the nucleic acid is determined from the sequence of releasednucleotides. Various exonucleases known in the art are useful forexonuclease sequencing, including but not limited to T7 exonuclease,ExoIII, ExoVII, mung bean nuclease, lambda exonuclease, and theexonuclease activity of various polymerases (e.g., Klenow, poll, Taqpolymerase, and T4 polymerase). Sequencing by exonuclease degradation isdescribed further, e.g., in U.S. Pat. Nos. 5,622,824 and 5,516,633; andin international application no. PCT/US1994/003416. A template nucleicacid immobilized by a template localizing moiety can be subjected todegradation by an exonuclease and the resulting free nucleotides can bedetected by methods known in the art, including mass spectrometry,optical detection of fluorescent or luminescent labels on the releasednucleotides, passage through a nanopore, etc.

In further embodiments, a combination of an exonuclease and a polymerasecan be used to determine the sequence of a template nucleic acid, e.g.,by subjecting a single-stranded circular template nucleic acid torolling circle amplification by the polymerase, degrading the resultingnascent strand with an exonuclease, and detecting the release ofnucleotides. This method provides an added benefit by allowing repeatedsequencing of the circular template since the exonuclease acts only onthe nascent strand.

FURTHER DETAILS REGARDING NUCLEIC ACID AMPLIFICATION AND SEQUENCING

The compositions of the invention, e.g., surface-immobilized templatelocalizing moieties, can be used in combination with sequencing enzymeto sequence a template nucleic acid. In certain embodiments, thesequencing enzyme can associate with the template localizing moiety in anon-covalent manner or bind the moiety via a reversibly cleavablelinker, e.g., a linker that can reform with a new sequencing enzyme.Thus, the template can advantageously be sequenced in a manner thatpermits the exchange of a first, e.g., inactive, sequencing enzyme, witha second, e.g., active, sequencing enzyme, without disrupting thesequencing reaction. for example, during template-dependent synthesis ofa nascent nucleic acid, an inactive polymerase can be replaced by anactive polymerase, allowing stalled nascent strand synthesis toreinitiate. In other embodiments of the sequencing reactions provided bythe invention, a sequencing enzyme can be covalently bound to theimmobilized template localizing moiety, e.g., at the C-terminal end of apolymerase (see, e.g., FIG. 4).

The template nucleic can be a linear or circular molecule, and incertain applications, is desirably a circular template (e.g., forrolling circle replication or for sequencing of circular templates), asshown in FIGS. 2 and 3. Optionally, the composition can be present in anautomated nucleic acid synthesis and/or sequencing system. A templatenucleic acid can be double-stranded or single-stranded, and can compriseDNA, RNA, analogs and/or derivatives thereof, and combinations of thesame. A template nucleic acid can comprise chemical modifications (e.g.,labels, nucleotide analogs or derivatives, etc.).

For template-directed sequencing-by-synthesis reactions, a replicationinitiating moiety in the reaction mixture can be a standardcomplementary oligonucleotide primer, or, alternatively, a component ofthe template, e.g., the template can be a self-priming single-strandedDNA, a nicked double-stranded DNA, or the like. Such an oligonucleotideprimer can comprise native or modified nucleotides, or derivatives,analogs, and/or combinations thereof. Similarly, a terminal protein canserve as an initiating moiety. At least one nucleotide analogue can beincorporated into the DNA. Additional details of and methods forsequencing by incorporation methods are known in the art, e.g., in U.S.Pat. Nos. 6,787,308, 6,255,083, 5,547,839, and 6,210,896; U.S.S.N.2004/0152119, 2003/0096253, 2004/0224319, 2004/0048300, 2003/0190647,and 2003/0215862; and international application nos. WO/1996/027025,WO/1999/005315, and WO/1991/006678, all of which are incorporated hereinby reference in their entireties for all purposes.

The compositions of the invention can localize the incorporation oflabeled nucleotides/analogs to a defined reaction region. This can be ofparticularly beneficial use in a variety of different nucleic acidanalyses, including real-time monitoring of DNA polymerization anddegradation. For example, a fluorescent or chemiluminescent label can beincorporated, or more preferably, can be released during incorporationof the analogue into a nascent nucleic acid strand. For example,analogue incorporation can be monitored in real-time by monitoring labelrelease during incorporation of the analogue by a polymerase that canexchange with a second polymerase in the reaction mixture, e.g., withoutterminating the sequence read. The portion of a nucleotide analogue thatis incorporated, e.g., into the copied nucleic acid can be the same as anatural nucleotide, or can include features of the analogue that differfrom a natural nucleotide. Alternatively or additionally, other methodsfor detection of nucleotide incorporation may be employed, e.g.,luciferase-mediated detection of released pyrophosphate.

In general, label incorporation or release can be used to indicate thepresence and composition of a growing nucleic acid strand, e.g.,providing evidence of template-directed synthesis/amplification and/orsequence of the template. Signaling from the incorporation can be theresult of detecting labeling groups that are liberated from theincorporated analogue, e.g., in a solid phase assay, or can arise uponthe incorporation reaction. For example, in the case of FRET labelswhere a bound label is quenched and a free label is not, release of alabel group from the incorporated analogue can give rise to afluorescent signal. Alternatively, polymerases present in a sequencingreaction mixture, e.g., that can be exchanged during the sequencingreaction, may be labeled with one member of a FRET pair proximal to theactive site, and incorporation of an analogue bearing the other memberwill allow energy transfer upon incorporation. The use of enzyme boundFRET components in nucleic acid sequencing applications is described,e.g., in U.S. Patent Application Publication No. 2003/0044781,incorporated herein by reference.

In one example reaction of interest, a surface-bound template localizingmoiety can be used to isolate a nucleic acid polymerization reactionwithin an extremely small observation volume that effectively results inobservation of individual template-directed synthesis reactions. As aresult, the incorporation event provides observation of an incorporatingnucleotide analogue that is readily distinguishable fromnon-incorporated nucleotide analogues. That is, when a polymeraseincorporates complementary, fluorescently labeled nucleotides into thenucleic acid strand that is being synthesized, the enzyme holds eachnucleotide within the detection volume for tens of milliseconds, e.g.,orders of magnitude longer than the amount of time it takes anunincorporated nucleotide to diffuse in and out of the detection volume.As described above, the polymerase can be exchanged with a secondpolymerase in the reaction mixture without terminating the sequence ofincorporation events.

In a preferred aspect, such small observation volumes are provided byimmobilizing the template localizing moiety within an opticalconfinement, such as a Zero Mode Waveguide (ZMW). For a description ofZMWs and their application in single molecule analyses, and particularlynucleic acid sequencing, see, e.g., U.S. Patent Application PublicationNo. 2003/0044781, and U.S. Pat. No. 6,917,726, each of which isincorporated herein by reference in its entirety for all purposes. Seealso Levene et al. (2003) “Zero-mode waveguides for single-moleculeanalysis at high concentrations” Science 299:682-686 and U.S. Pat. Nos.7,056,676, 7,056,661, 7,052,847, and 7,033,764, the full disclosures ofwhich are incorporated herein by reference in their entirety for allpurposes. Although various embodiments of the invention are describedprimarily in terms of zero-mode waveguide substrates, other types ofsubstrates comprising appropriately configured reaction regions areknown in the art and useful with the methods, compositions, and systemsdescribed herein, including but not limited to waveguide substrates,TIRE substrates, and the like. See, e.g., U.S. Patent Publication No.20080128627; and U.S. Ser. No. 61/192,326, filed Sep. 16, 2009, both ofwhich are incorporated herein by reference in their entireties for allpurposes.

A surface-immobilized template localizing moiety that fixes the templatestrand within, e.g., a ZMW, in the presence alone or more nucleotidesand/or one or more nucleotide analogues, e.g., fluorescently labelednucleotides or nucleotide analogs. For example, in certain embodiments,labeled analogues are present representing analogous compounds to eachof the four natural nucleotides, A, T, G and C, e.g., in separatepolymerase reactions, as in classical Sanger sequencing, or multiplexedtogether, e.g., in a single reaction, as in multiplexed sequencingapproaches. When a particular base in the template strand is encounteredby a polymerase during the polymerization reaction, it complexes with anavailable analogue that is complementary to such nucleotide, andincorporates that analogue into the nascent and growing nucleic acidstrand. In one aspect, incorporation can result in a label beingreleased, e.g., in polyphosphate analogues, cleaving between the α and βphosphorus atoms in the analogue, and consequently releasing thelabeling group (or a portion thereof). The incorporation event isdetected, either by virtue of a longer presence of the analogue and,thus, the label, in the complex, or by virtue of release of the labelgroup into the surrounding medium. Where different labeling groups areused for each of the types of analogues, e.g., A, T, G or C,identification of a label of an incorporated analogue allowsidentification of that analogue and consequently, determination of thecomplementary nucleotide in the template strand being processed at thattime. Sequential reaction and monitoring permits a real-time monitoringof the polymerization reaction and determination of the sequence of thetemplate nucleic acid.

As noted above, in particularly preferred aspects, the templatelocalizing moiety, e.g., that is configured to interact with apolymerase, is provided immobilized within an optical confinement thatpermits observation of an individual template-dependent synthesisreaction in, e.g., a Zero-Mode Waveguide. An immobilized templatelocalizing moiety can fix a template to a surface, beneficially providelonger and more accurate sequence reads in that, e.g., a polymerase thathas sustained photodamage as a result of exposure to the optical energyof the fluorescently labeled nucleotides or nucleotide analogues presentin the reaction mix can exchange with, e.g., a non-photodamagedpolymerase, during a template-dependent polymerization reaction.

In addition to their use in sequencing, the surface-immobilized templatelocalizing moieities of the invention are also useful in a variety ofother analyses, e.g., real time monitoring of amplification, e.g.,real-time-PCR methods, and the like. For example, real-time nucleicamplification reactions that include one or very few nucleic acidtemplate molecules can be performed more efficiently if the template andpolymerase were co-localized, e.g., by surface-immobilized templatelocalizing moiety, e.g., that has been configured to interact with apolymerase. Further details regarding sequencing and nucleic acidamplification can be found, e.g., Berger and Kimmel, Guide to MolecularCloning Techniques. Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 2001 (“Sambrook”); Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc (“Ausubel”); Kaufman et al. (2003) Handbook of Molecular andCellular Methods in Biology and Medicine Second Edition Ceske (ed) CRCPress (Kaufman); and The Nucleic Acid Protocols Handbook Ralph Rapley(ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley).

FURTHER DETAILS REGARDING INTEGRATION OF METHODS/COMPOSITIONS INTO HIGHTHROUGHPUT SEQUENCING SYSTEMS

The methods and compositions provided by the invention canadvantageously be integrated with systems that can, e.g., automateand/or multiplex the sequencing reactions comprising asurface-immobilized template localizing moiety. Systems of the inventioncan include one or more modules, e.g., that automate a method herein,e.g., for high-throughput sequencing applications. Such systems caninclude fluid-handling elements and controllers that move reactioncomponents into contacts with one another, signal detectors, systemsoftware/instructions, e.g., to convert a sequence of fluorescentsignals into nucleotide sequence information, and the like.

Systems provided by the invention include a reaction region in which atemplate localizing moiety has been immobilized, e.g., with a covalentbond. The template localizing moiety in the reaction region canoptionally be configured to interact with a sequencing enzyme, e.g., anyone of the sequencing enzymes described herein. The one or moresingle-molecule reaction region of the system can optionally include asequencing enzyme, which, in certain embodiments of the systems, can becovalently linked to the surface-immobilized template localizing moiety,e.g., via a polymerase's C-terminal end (see FIG. 4) or linked, e.g.,via a reversibly cleavable linker, e.g., a linker that can reform with anew sequencing enzyme.

In preferred embodiments, the sequencing enzyme can form a non-covalentcomplex with the template localizing moiety in the reaction region suchthat the sequencing enzyme can exchange with a second sequencing enzymepresent, e.g., in a reaction mixture, without interrupting thesequencing reaction. This can beneficially provide longer and moreaccurate sequence reads in that, e.g., a sequencing enzyme that hassustained photodamage as a result of exposure to the optical energy ofthe fluorescently labeled nucleotides or nucleotide analogues present inthe reaction mix can exchange with, e.g., a non-photodamaged sequencingenzyme, during a sequencing reaction.

The reaction region can optionally comprise a planar surface, well, orone or more single-molecule reaction region. In preferred embodiments,the reaction region can optionally comprise one or more Zero ModeWaveguides (ZMWs). (See, e.g., Levene et al. (2003) “Zero-modewaveguides for single-molecule analysis at high concentrations” Science299:682-686 and U.S. Pat. Nos. 7,056,676, 7,056,661, 7,052,847, and7,033,764, the full disclosures of which are incorporated herein byreference in their entirety for all purposes.)

Systems of the invention can optionally include modules that provide fordetection or tracking of products, e.g., a fluorescent light from one ormore fluorophore that is linked to a nucleotide or nucleotide analogthat is being incorporated into a growing nucleic acid. Detectors caninclude spectrophotometers, epifluorescent detectors, CCD arrays, CMOSarrays, microscopes, cameras, or the like. Optical labeling isparticularly useful because of the sensitivity and ease of detection ofthese labels, as well as their relative handling safety, and the ease ofintegration with available detection systems (e.g., using microscopes,cameras, photomultipliers, CCD arrays, CMOS arrays and/or combinationsthereof). High-throughput analysis systems using optical labels includeDNA sequencers, array readout systems, cell analysis and sortingsystems, and the like. For a brief overview of fluorescent products andtechnologies see, e.g., Sullivan (ed) (2007) Fluorescent Proteins,Volume 85, Second Edition (Methods in Cell Biology) (Methods in CellBiology) ISBN-10: 0123725585; H of et al. (eds) (2005) FluorescenceSpectroscopy in Biology: Advanced Methods and their Applications toMembranes, Proteins, DNA, and Cells (Springer Series on Fluorescence)ISBN-10: 354022338X; Haughland (2005) Handbook of Fluorescent Probes andResearch Products, 10th Edition (Invitrogen, Inc./Molecular Probes);BioProbes Handbook, (2002) from Molecular Probes, Inc.; and Valeur(2001) Molecular Fluorescence: Principles and Applications WileyISBN-10: 352729919X. System software, e.g., instructions running on acomputer can be used to track and inventory reactants or products,and/or for controlling robotics/fluid handlers to achieve transferbetween system stations/modules. The overall system can optionally beintegrated into a single apparatus, or can consist of multiple apparatuswith overall system software/instructions providing an operable linkagebetween modules.

Kits

The present invention also provides kits that incorporate thecompositions of the invention. Such kits can include, e.g., a templatelocalizing moiety packaged in a fashion to permit its covalent bindingto a surface of interest. Alternatively the surface bound templatelocalizing moieties can be provided as components of the kits, or thesurface can be provided with binding partners suitable to bind thetemplate localizing moieties, which are optionally packaged separately.Instructions for making or using surface bound template localizingmoieties are an optional feature of the invention.

The template localizing moieties provided in such kits can also comprisepolynucleotide complementary to a polynucleotide sequence of interest ina template nucleic acid to facilitate selective immobilization of asubset of template nucleic acids having one or more particularpolynucleotide sequences of interest (e.g., exonic or intronic regions,regulatory regions, and the like). For example, a kit can comprise apool of template localizing moeties having polynucleotide regionscomplementary to a set of genetic loci known to predict susceptibilityto a given disease, identify an unknown microorganism, determinepaternity, and other forensic, medical, and agricultural analyses. Onlygenomic fragments having one or more of those genetic loci of interestwill be targeted and immobilized by the template localizing moieties,and subsequently subjected to sequence analysis, thereby allowingselective analysis of a subset of a complex genomic sample and areduction in the complexity of the data set so generated.

Such kits can also optionally include additional useful reagents such asone or more nucleotide analogs, e.g., for sequencing, nucleic acidamplification, or the like. For example, the kits can include asequencing enzyme packaged in such a manner as to enable its use withthe template localizing moiety, a set of different nucleotide analogs ofthe invention, e.g., those that are analogous to A, T, G, and C, e.g.,where one or more of the analogs comprise a detectable moiety, to permitidentification in the presence of the analogs. The kits of the inventioncan optionally include natural nucleotides, a control template, andother reagents, such as buffer solutions and/or salt solutions,including, e.g., divalent metal ions, i.e., Mg⁺⁺, Mn⁺⁺ and/or Fe⁺⁺,standard solutions, e.g., dye standards for detector calibration, etc.Such kits also typically include instructions for use of the compoundsand other reagents in accordance with the desired application methods,e.g., nucleic acid sequencing, nucleic acid labeling, amplification,enzymatic detection systems, and the like.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed is:
 1. A method of sequencing a nucleic acid, the methodcomprising: fixing a template nucleic acid to a solid surface through atemplate localizing moiety, wherein the template localizing moietytopologically encircles the template such that the template can movethrough the template localizing moiety; sequencing a portion of at leastone strand of the template nucleic acid with a first sequencing enzyme;exchanging the first sequencing enzyme with a second sequencing enzyme;and, continuing sequencing of the strand with the second sequencingenzyme.
 2. The method of claim 1, wherein the first sequencing enzyme isa first polymerase, the second sequencing enzyme is a second polymerase,and the template nucleic acid is, a circular template nucleic acid. 3.The method of claim 2, further comprising sequencing the templatenucleic acid multiple times with a plurality of polymerases to generatea single nucleic acid strand comprising multiple copies of apolynucleotide complementary to the template nucleic acid.
 4. A methodof sequencing a template nucleic acid, the method comprising: fixing acircular template nucleic acid to a solid surface through a templatelocalizing moiety, wherein the template localizing moiety topologicallyencircles the template such that the template can move through thetemplate localizing moiety; annealing an oligonucleotide primer to thetemplate nucleic acid; initiating template-directed nascent strandsynthesis by a polymerase that is not immobilized to the solid surface;synthesizing a nascent strand complementary to the template nucleic acidwith the polymerase detecting incorporations of nucleotides into thenascent strand, wherein a temporal sequence of the incorporations isindicative of the sequence of the nucleic acid.
 5. The method of claim4, further comprising sequencing the template nucleic acid multipletimes to generate a single nascent stand comprising multiple copies of apolynucleotide complementary to the template nucleic acid.
 6. The ofclaim 4, wherein the polymerase is a plurality of polymerase enzymes,and further wherein only a single of the plurality is engaged in thetemplate-directed nascent strand synthesis on the template nucleic acidat a given time.
 7. The method of claim 1, wherein exchanging the firstsequencing enzyme with the second sequencing enzyme comprises exchanginga photodamaged sequencing enzyme with a non-photodamaged sequencingenzyme.
 8. The method of claim 1, wherein sequencing is continued withthe second sequencing enzyme, wherein the second sequencing enzyme isnon-photodamaged.
 9. The method of claim 4, the nucleotides comprisedetectable labels that identify the base composition of the nucleotides.10. The method of claim 4, wherein the incorporations are detected usinga luciferase-mediated detection system.
 11. The method of claim 4,wherein template nucleic acid is a single-stranded template nucleicacid.
 12. The method of claim 1, wherein the template localizing moietycomprises a synthetic polymer.
 13. The method of claim 4, wherein thetemplate localizing moiety comprises a synthetic polymer.
 14. The methodof claim 1, wherein the template localizing moiety is asurface-immobilized template localizing moiety.
 15. The method of claim4, wherein the template localizing moiety is a surface-immobilizedtemplate localizing moiety.